Methods for ms2 quantitation of isobaric labeled compounds

Abstract

A method for correcting abundance ratios between pairs of isobaric reporter ions comprises: (a) measuring, for each liberated reporter-ion moiety the variation, with time, of a signal from said moiety; (b) identifying a first set and a second set of reporter-ion moieties for which the respective signal is, respectively, positively correlated with and not correlated with, the time variation of one or more other signals or variables that pertain to the detection of one or more peptides of interest; (c) for each reporter-ion moiety, decomposing the respective measured mass spectrometric signal into first and second portions that, respectively are and are not attributable to the peptide; (d) for each identified reporter-ion moiety, setting a respective adjusted mass spectrometric signal as being the respective portion of the signal that is attributable to the peptide; and (e) calculating corrected reporter-ion ratios based on the adjusted mass spectrometric signals.

What is claimed is: 
     
         1 . A method for correcting abundance ratios between reporter ions detected during tandem mass analysis (MS2) of a sample that comprises a plurality of peptides that are labeled with a plurality of different isobaric tags, each isobaric tag comprising a respective reporter-ion moiety that comprises a respective predetermined reporter-ion formula weight and a respective mass-balancing moiety that comprises a respective predetermined mass-balancing formula weight, the method comprising:
 during a time period, measuring, for each reporter-ion moiety liberated by the tandem mass analysis, the variation, with time, of a respective mass spectrometric signal derived from said respective reporter-ion moiety;   identifying a first set of reporter-ion moieties for which the respective mass spectrometric signal is positively correlated, with an above-threshold correlation coefficient, with the time variation, during the time period, of one or more other mass spectrometric signals, parameters or variables that pertain to the detection of a peptide of interest and identifying a second set of reporter-ion moieties for which the respective mass spectrometric signal is not positively correlated with the one or more other mass spectral signals or variables;   based on the correlations, for each identified reporter-ion moiety of the first set, decomposing the respective measured mass spectrometric signal into a first portion that is attributable to the peptide of interest and a second portion that is not attributable to the peptide of interest;   for each identified reporter-ion moiety, setting a respective adjusted mass spectrometric signal as being the respective portion of the mass spectrometric signal that is attributable to the peptide of interest; and   calculating corrected reporter-ion ratios based on the adjusted mass spectrometric signals.   
     
     
         2 . A method as recited in  claim 1 , wherein the decomposing of each measured mass spectrometric signal into a respective first portion that is attributable to the peptide, and a second portion that is not attributable to the peptide comprises calculating the first and second portions using a non-negative least squares analysis. 
     
     
         3 . A method as recited in  claim 1 , wherein the time variation of the one or more other mass spectrometric signals, parameters or variables that pertain to the detection of one or more peptides of interest are determined from tabulated values of peptide elution times and/or elution profiles. 
     
     
         4 . A method as recited in  claim 1 , wherein the time variation of the one or more other mass spectrometric signals, parameters or variables that pertain to the detection of one or more peptides of interest are determined by measuring the variation of the one or more other mass spectrometric signals or variables during the time period. 
     
     
         5 . A method as recited in  claim 4 , wherein the one or more other mass spectrometric signals, parameters or variables that are measured during the time period comprise mass spectra of characteristic peptide fragment ions. 
     
     
         6 . A method as recited in  claim 1 , wherein the beginning of the time period is triggered by a time delay relative to the beginning of an experiment. 
     
     
         7 . A method as recited in  claim 1 , wherein the tandem mass analysis of the sample comprises a data-dependent analysis (DDA) experiment, wherein the beginning of the time period is triggered by detection of an m/z value, within an MS1 mass spectrum, that corresponds to a peptide of interest. 
     
     
         8 . A method as recited in  claim 1 , wherein the tandem mass analysis of the sample comprises a data-dependent analysis (DDA) experiment, wherein the beginning of the time period is triggered by the prior steps of:
 detection of a first m/z value, within an MS1 mass spectrum, that corresponds to a potential peptide of interest;   automatic consultation of a mass spectral library to determine a second m/z value to be searched for within a subsequent MS2 mass spectrum;   generation of fragment ions by fragmentation of ions comprising the first m/z value; and   detection of the second m/z value within an MS2 spectrum of the fragment ions.   
     
     
         9 . A method as recited in  claim 1 , wherein the tandem mass analysis of the sample comprises a data-independent analysis (DIA) experiment, wherein the time period is one of a plurality of scheduled overlapping time periods. 
     
     
         10 . A method as recited in  claim 1 , wherein the beginning of the time period is triggered by elapse of an ion mobility drift time subsequent to the introduction of a stream of peptide ions labeled with the plurality of different isobaric tags into an ion mobility separation device that receives the ions from an ion source and that delivers ion mobility separated ions to a mass spectrometer that performs the tandem mass analysis. 
     
     
         11 . A method as recited in  claim 1 , wherein the variation, with time, of the mass spectrometric signal derived from each respective reporter-ion moiety and time variation of one or more other mass spectrometric signals or variables is at least partially caused by time variation of a compensation voltage (CV) applied to a Field Asymmetric Ion Mobility Separation (FAIMS) device that that receives ions from an ion source and that delivers ion mobility separated ions to a mass spectrometer that performs the tandem mass analysis. 
     
     
         12 . A method as recited in  claim 1 , wherein the variation, with time, of the mass spectrometric signal derived from each respective reporter-ion moiety and time variation of one or more other mass spectrometric signals or variables is at least partially caused by time variation of one or more operational parameters of a Trapped Ion Mobility Separation (TIMS) device that receives ions from an ion source and that delivers ion mobility separated ions to a mass spectrometer that performs the tandem mass analysis. 
     
     
         13 . A mass spectrometer system comprising:
 an ion source that generated first-generation ions from a sample;   an ion trap or reaction cell configured to receive first-generation ions from the ion source;   a mass analyzer configured to receive a portion of the first-generation ions or fragment ions generated by fragmentation of a portion of the first-generation ions from the ion trap or reaction cell;   one or more electrical power supplies electrically coupled to the ion source, the ion trap or reaction cell and the mass analyzer; and   a computer processor electrically coupled to the ion source, the ion trap or reaction cell and the mass analyzer and comprising tangibly-embodied non-transitory computer instructions that are operable to cause the one or more power supplies to provide voltages or electrical signals to the ion source, the ion trap or reaction cell and the mass analyzer that cause the mass spectrometer system to execute the method of  claim 1 .   
     
     
         14 . A mass spectrometer system as recited in  claim 13 , further comprising:
 a computer-readable medium that comprises an input database and that is electrically coupled to the computer processor,   wherein the tangibly-embodied non-transitory computer instructions are further operable to receive information from the input database and to cause the one or more power supplies to provide voltages or electrical signals to the ion source, the ion trap or reaction cell and the mass analyzer that cause the mass spectrometer system to execute the method of  claim 8 .   
     
     
         15 . A mass spectrometer system as recited in  claim 13 , further comprising:
 a Field Asymmetric Ion Mobility Separation (FAIMS) device fluidically coupled between the ion source and the ion trap or reaction cell,   wherein the tangibly-embodied non-transitory computer instructions are further operable to provide voltages or electrical signals to the ion source, the ion trap or reaction cell, the FAIMS device and the mass analyzer that cause the mass spectrometer system to execute the method of  claim 11 .   
     
     
         16 . A mass spectrometer system as recited in  claim 13 , further comprising
 a Trapped Ion Mobility Separation (TIMS) device fluidically coupled between the ion source and the ion trap or reaction cell,   wherein the tangibly-embodied non-transitory computer instructions are further operable to provide voltages or electrical signals to the ion source, the ion trap or reaction cell, the TIMS device and the mass analyzer that cause the mass spectrometer system to execute the method of  claim 12 .